Tapai is a well-known indigenous fermented alcoholic beverage among Kadazan-Dusun-Murut (KDM) ethnics during
festive occasions and gatherings in East Malaysia. Unfortunately, very little research has been done on this beverage.
The objective of this study was to identify functional microfloras involved in the production of tapai. Samples from local
producers were obtained for microbiological and proximate analysis. The fermentation process was predominated by
yeasts and lactic acid bacteria (LAB) with initial numbers (CFU/g) of 105
and 106
, respectively, which gradually increased
during the first 2 weeks fermentation but decreased thereafter. The yeasts were identified as Sacchromyces cerevisiae,
Candida krusei, C. pelliculosa, C. guillermondii, C. magnoliae and Rhodotorula glutinis, whereas the LAB were
Lactobacillus brevis, L. plantarum, L. collinoides and Pediococcus sp. Moulds and Enterobacteriaceae were only present
during the first 2 days of fermentation. Acetic acid bacteria were not detected throughout the entire process. The pH of
tapai declined slowly from 6.6 to 3.4 in 14 days, and then showed an increment to 4.0. On the other hand, titratable
acidity (as % lactic acid) increased from 0.06 to 0.86 in 10 days, and then decreased to 0.82 at the end of the
fermentation process. Alcohol was produced and the content can reach as high as 12.3% after 3 weeks fermentation.
Proximate composition analysis showed that the moisture content in the end product was 61.8±6.1% whereas ash,
protein, fat and crude fiber (of dried samples) were 0.50±0.1%, 8.7±0.1%, 0.29±0.01% and 0.56±0.03%, respectively.
Keywords: Microflora, Indigenous, Fermented beverage, Malaysia

INTRODUCTION
Indigenous fermented foods have become a new interest
and consequently provided new subjects for intellectual
creation these few years. While traditionally produced
food products may be of health concern to non traditional
consumers due to the therapeutic properties of fermented
food reported, advanced scientific knowledge on food
fermentation and its microbial agent has increasingly
revealed many beneficial effects which lead to new
applications other than food preservation, safety and
sensory appreciation. Many studies have been done on
indigenous fermented foods from around the world
(Tamang and Thapa, 2004; Sefa-Dedeh et al., 2004;
Mugula et al., 2003; Muyanja et al., 2002; Leisner et al.,
2001; Omafuvbe et al., 2000; Wacher et al. 2000) and
information on microbiological, biochemical and nutritional
changes during fermentation is well documented.
Moreover, products like Indonesian tempe and Oriental
soy sauce are well known indigenous fermented foods
that have industrialized and marketed globally years ago
(Wood, 1994).

Tapai is a well-known indigenous fermented alcoholic
beverage among the Kadazan-Dusun-Murut (KDM) ethnic
group of Sabah during festive occasions and gatherings.
Unlike tapai in other Southeast Asia countries like tape
ketan (Indonesia), tapai (Peninsular Malaysia, Singapore
and Brunei), basi (Philipines) and Khao-mak (Thailand) (Campbell-Platt, 2000) which are prepared as a food,
Sabah’s tapai is prepared as an alcoholic beverage. It has
an alcoholic aroma with combination of sweet-sour-bitter
taste and sometimes sparkling feel. Tapai is made from
glutinous rice with sasad as starter culture although rice,
cassava, pineapples and maize can be used as substitute
for glutinous rice in some part of Sabah. During
preparation of tapai, glutinous rice is cleaned and washed
before cooked. Cooked glutinous rice is then spread for
cooling in an open surface (≈30oC). Starter culture cakes
are grinded into powder and approximately 1.0-1.5% (by
weight) is sprinkled on the cooled glutinous rice followed
by mixing thoroughly using a wooden scoop. The mixture
is transferred into tajau (earthen jar) and left open for 1
day before the lid of tajau is sealed. Good quality and
matured tapai undergoes 3 weeks fermentation. There are
few ways to consume tapai. The most popular way is by
drinking hing or lihing (wine must). Tapai can also be
consumed as kinomulok (fermented glutinous rice after
the wine must have been separated) and linutau (water
extract from kinomulok). Siopon or Sisopon is a way of
consumption where a thin bamboo straw is inserted into
water added kinomol (fermented glutinous rice in tajau) for
sipping tapai extracts. Other than that, Montoku and talak
(distilled wine must) are famous alcoholics beverage
among KDM.

Unfortunately at present, there is no adequate
information on the spectrum of microorganisms
associated with tapai fermentation in Sabah as information on indigenous fermented food is extremely
rare although this knowledge is essential for the
development of this product with improved quality for
commercial production and marketing. Thus, the objective
of this research was to identify functional microfloras
involved in the microbiological, biochemical and nutritional
changes that took place during tapai production.

MATERIALS AND METHODSEnumeration, isolation and identification
Freshly inoculated glutinous rice in tajau were purchased
from a local tapai producer in Kampung Karanaan,
Tambunan, Sabah and transported to the Food
Microbiology Laboratory of the School of Food Science
and Nutrition at University Malaysia Sabah for immediate
microbial analysis. Twenty five grams of samples were
homogenized with 225 ml of Quarter Strength Ringer’s
Solution (Merck, Germany) in a Bag Mixer (Interscience,
France) for 30 min and serially diluted in the same diluent
in duplicate. During tapai fermentation, the same amount
of sample was collected from the tajau for microbial
analysis every 2 successive days for 20 days. Yeast and
mould counts were determined using Dichloran Rose
Bengal Choramphenicol (DRBC) Agar (Merck, Germany)
(Ardhana & Fleet, 2003) and incubated at 25oC for 3 days.
Lactic acid bacteria (LAB) were enumerated in M17 Agar
(Merck, Germany) aerobically and on de Man Rogosa and
Sharpe (MRS) Agar (Merck, Germany) anaerobically
(Oxoid Carbon Dioxide System BR 39, England) at 30oC
for 2 days (Mugula et al., 2003). Acetic acid bacteria were
enumerated using Glucose Yeast Extract Calcium
Carbonate Agar (glucose 5%, yeast extract 1%, calcium
carbonate 3% and agar 2%) and incubated at 30oC for 6
days (Du Toit and Lambrechts, 2002). Enterobacteriaceae
and aerobic mesophilic count was determined using Violet
Red Bile Glucose Agar and Plate Count Agar (Merck,
Germany) (Thapa and Tamang, 2004) at 37oC for 2 days,
respectively.

About 10-15 colonies were selected randomly from
the plates. Purity of the isolates was checked by streaking
again on fresh agar plates of the isolation medium. Yeasts
and LAB isolates were stored at Potato Dextrose Agar
(Merck, Germany) and MRS Agar slants, respectively.
Yeasts were identified according to their cell morphology,
physiological, carbon sources assimilation and
fermentation patterns described by Kurtzman and Fell
(1998) supplemented with API 20C AUX test strips
(BioMérieux, France). LAB were identified according to
cell and colony morphology, Gram and catalase reactions,
gas production from glucose and fermentation pattern in
API 50 CH and API 50 CHL Medium (BioMérieux,
France).

Analysis of proximate composition
Ten grams sample of fermented glutinous rice was
blended with 20 ml of distilled water in a homogenizer for
30 seconds and the pH of the slurry was determined by
digital pH meter calibrated with standard buffer solutions
(Merck, Germany). Titratable acidity (expressed as
percent lactic acid) of tapai was determined by titrating the
filtrates, a well blended 10 g sample in 90 ml distilled
water with 0.1 N sodium hydroxide to end point using
phenolphthalein as indicator. Alcohol content was
determined by blending 10 g sample with 90 ml distilled
water in the homogenizer described above using
distillation method.

The moisture content was determined by drying
samples in oven for overnight at 70oC to constant weight.
Ash content was determined by heating the dried end
product in furnace at 550oC overnight till the difference
between two successive weighing was not more than
0.1%. Protein and fat contents were determined using
Kjeldahl distillation method and Soxhlet method on dried
sample respectively. For crude fiber content, 2 g sample
was boiled for 30 minutes in 12.5% sulfuric acid, filtered
and washed with hot distilled water untill no longer acidic
before boiling in 12.5% sodium hydroxide and the same
procedure repeated except it was filtered through a Gooch
crucible and washed till no longer alkaline. Crucible with
its content was dried at 100oC, transferred into the
incinerator oven and heated at 550oC overnight till the
difference between two successive weighing was not
more than 0.1%. Crude fiber content was estimated by
calculating percentage of weight loss in the sample.
Carbohydrate content of sample was estimated by
difference: 100-(%moisture + %protein + %fat + %ash +
%fiber) (Nitisewojo, 1995). The determinations were done
in triplicates and the mean values recorded.

RESULTS AND DISCUSSIONMicroorganisms
Tapai fermentation was predominated by yeast and LAB
as they were present from day 0 to day 20. Yeasts and
moulds grew to 8.0 log CFU/g on the 4th day of
fermentation from 5.1 log CFU/g but the population of
yeast population declined gradually thereafter until 6.1 log
CFU/g. However, moulds were undetected on the 5th day
onwards (Figure. 1). The population of LAB with 6.1 log
CFU/g initially became 1.0 log CFU/g greater on the 4th
day and decreased to 5.1 log CFU/g after 20 days. Acetic
acid bacteria was not detected in all tapai samples.
Enterobacteriaceae numbers declined from 4.6 log CFU/g
till an undetectable number within 4 days whereas aerobic
mesophilies increased from 6.6 log CFU/g to 8.4 log
CFU/g in 2 days and decreased to 5.3 log CFU/g at the
end fermentation process.

Saccharomyces cerevisiae, Candida krusei, C.
pelliculosa, C. glabrata, C. utilis, C. sphaerica, C.
magnoliae, Rhodotorula mucilaginosa, R. glutinis and
Cryptococcus laurentii were identified from among the
yeasts isolated (Table 1). S. cerevisiae occurred in the
highest number in tapai and was present at all stages of
the fermentation. It has been isolated frequently from
acidic fermentation of plant materials such as sourdough
(Gobetti et al., 1994) and ogi (Nago et al., 1998). Thus, it has been suggested that as playing a primary role in alcohol fermentation (Tsuyoshi et al., 2005). C.krusei, C. pelliculosa and C.glabrata were previously isolated from fufu (fermented cassava roots) (Oyewole, 2001), togwa (fermented sorghum, millet or finger millet) (Mugula et al., 2003) and kodo ko jaanr (alcoholic fermented finger millet beverage) (Thapa and Tamang, 2004), respectively and C.krusei has been associated with flavorand aroma of the end product of fufu (Oyewole, 2001). Saccharomyces and Candida species were reported capable of proliferating at low pH in porridge (Akinrele, 1970; Nout et al., 1989). C. utilis is known to produce a significant amount of ethyl acetate from biomass such as diluted sugar and ethanol. Ethyl acetate is recognized as an important flavor compound in wine and other grape derived alcoholic beverages. Such non-Saccharomyces yeast might contribute to flavor and aroma in the alcoholic beverages. C. sphaerica which was the anamorph state of K.lactis were isolated. K. lactis has found to be present in fermented dairy products such as cheese (Fadda et a., 2001) and is currently used for industrial applications for years as a source of beta-galactosidase (Bolotin-Fukuhara et al., 2000). Unsurprisingly C. magnoliae was food spoilage yeast and its presence may be due to contamination of the starter cake or utensil used during preparation as well as R. mucilaginosa and R. glutinis which were previously referred to as the species of common air contaminants or natural contaminants in cheese before they were stored (Viljoen and Greyling, 1995). Isolates not identified are due to inadequacy of the identification system used in the study and further investigation should be carried out. Though mould was only present in the early stage of fermentation, it was suspected to play a role in the degradation of glutinous rice into simpler substrate molecule to be utilized by yeast and LAB.

Eighty four percent strains of LAB were non-sporeforming rods (Table 1). They were tentatively identified as Lactobasillus plantarum, L. brevis and L. paracasei subsp. paracasei but only L. plantarum and L.brevis showed predomination in the tapai fermentation process and were isolated from at stages of fermentation. L.plantarum have been isolated from several indigenous fermented foods including togwa (Mugula et al., 2003), tempoyak (fermented durian fruit pulp) (Leisner et al., 2001) and kule nato (fermented milk) (Mathara, 2004). It has been recognized as the dominant organism at the end of several natural lactic acid fermentations (Brauman et al., 1996; Kunene et al., 2000), probably due to its acid tolerance (Fleming and McFeters, 1981) and superior ability to utilize the substrates (Oyewole and Odunfa, 1990). L. brevis often occur in fermenting plant material (Corsetti et al., 2001) and have been isolated from fermented foods like kenkey (fermented maize dough)
(Halm et al., 1993), mawe (fermented maize dough)
(Hounhouigan et al., 1993), and agbelima (fermented
cassava dough) (Kofi et al., 1996). L. paracasei spp.
paracasei has been reported to occur in boiled rice used
to prepare som fak, a Thai fermented fish (Paludan-Müller
et al., 1999). Sixteen percent of the LAB isolates were
cocci and identified as Pediococcus pentosaceus and
Lactococcus lactis subsp. lactis. Contrary to the report
which showed domination of P. pentosaceus in the latter
stage of corn dough fermentation (Nche et al., 1994), P.
pentosaceus was only present in the early stages of
fermentation, thus it seems that tapai fermentation is
initiated by P. pentosaceus but finally dominated by L.
plantarum as in the fermentation of mesu (fermented
bamboo shoot) (Tamang and Sarkar, 1996). Similar to P.
pentosaceus, Lactococcus lactis subsp. lactis were also
only present in the early stages of fermentation
correspond to the report by Muyanja et al. (2002) on
bushera due to its inability to grow at lower pH as the
tapai fermentation proceed.

A co-metabolism between yeasts and lactic acid
bacteria has been suggested, whereby the bacteria
provide the acid environment, which selects for the growth
of yeasts and, the yeasts provide vitamins and other
growth factors to the bacteria (Gobbetti et al., 1994;
Steinkraus, 1996). Yeasts have also been reported to
make a useful contribution to the improvement of flavour
and acceptability of fermented cereal gruels (Banigo et al.,
1974; Odunfa and Adeyele, 1985). Tapai is a safe
fermented product to consume as no enterobacteriaceae
were found at the end of the fermentation process,
probably due to its low pH, elevated titratable acidity and
high alcohol content. Their disappearance may also be
due to the presence of other antimicrobial compounds.
The absence of acetic acid bacteria in tapai could be due
to the sealed earthen jar which provide unsuitable growth
condition as they as strictly aerobic bacteria.

Proximate composition
The pH of inoculated glutinous rice was 6.6 initially and it
decreased rapidly and stabled steadily at 3.4 on the 15th
day. It increased to 4.0 at the end of fermentation. This
may be due to the increased of titratable acidity
(expressed as percent of lactic acid) from 0.06 to 0.86% in
10 days, and decreased to 0.82% in the end of tapai
fermentation (Figure 2). The correlation between acidity
and pH is believed to be associated with both yeasts and
LAB as LAB were well known for production of acids
especially lactic whereas some yeasts were previously
reported to produce acid in alcohol fermentation to make a
positive contribution to the products’ flavour (Fleet, 2003).
At the same time, low pH and high acidity also eliminated
enteropathogen, coliforms and spoilage organisms in this
product.

Meanwhile, its alcohol content increased day by day
and was as high as 12.3% (v/v) after 20 days fermentation
(Figure 3). Yeasts may produce alcohol; however,
Lactobacillus species have also been reported to produce
ethanol. Alcohols produced play a role in helping to
extract flavour components from fermenting substrates.
Yeasts and LAB also produce other volatile compounds
such as malty flavored 2-methyl-propanal. The flavors
contributed by yeasts and LAB yield unique fermented
products appreciated by consumers which were much
different from the unfermented substrate.

The moisture and ash contents in the end product of
tapai fermentation were 61.8±6.1% and 0.2±0.041%
respectively. Crude protein, crude fat and crude fiber (of
dried samples) were 3.3±0.04%, 0.1±0.004% and 0.2±0.01%, respectively. The estimated carbohydrate
content in tapai was 34.3±0.04%. Because of its high
calorie, tapai were not only consumed in festive occasions
and gatherings, but also by ailing persons and post natal
women to regain strength. Tapai were also consumed as
a family staple food by some ethnic groups in rural areas.
The result of this study indicated that tapai contains
a variety of yeasts and LAB. Various flavor compounds
were believed present in tapai making it a favorite
alcoholic beverage by Sabah peoples. Thus, controlled
fermentation should be done to assess contribution of
yeasts and LAB on flavor and aroma of this traditional
alcoholic fermented beverage. There is a need for
investigation into the selection of the most suitable strain
for better controlled tapai fermentation. Starter cultures
development is important for the potential small-scale
commercial production of tapai and for improvement of its
acceptability, microbiological stability and hygiene safety.
Detail avaibility of nutrient values which included minerals
and vitamins in tapai should be carried out. Strains
isolated from tapai could be screened for their properties
of exo- and endocellular enzymatic activities as well as
potential probiotic and nutraceutical properties for
application in improvement of human health.